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  1. 1. 3.19.2010<br /><ul><li>Small G proteins are single monomer proteins that bind GDP or GTP, GEFS help exchange GDP for GTP. GAPs enhance inactivation state on small g proteins, examples include… all regulated like a switch.
  2. 2. Ran
  3. 3. Rac
  4. 4. Rho
  5. 5. Cdc42
  6. 6. Heterotrimeric G Proteins
  7. 7. Alpha – Binds GTP, or GDP, and will hydrolyze GTP to GDP
  8. 8. Beta
  9. 9. Gamma
  10. 10. Bind to serpentine 7 receptors, which activate heterotrimeric G proteins
  11. 11. Embedded into membrane
  12. 12. Cytosolic side of receptor is the binding site for heterotrimeric g proteims, which contains a loop between strands 5 and 6 and ligand binding domain is between 6 and 7. – extracellular
  13. 13. When ligand binds to it, it exposes an activation site (binding site) for heterotrimeric g proteins, which is linked to the membrane (meristic acid that links protein to membrane) and when exposed, interacts with HTGP
  14. 14. Inactive G Proteins (alpha beta gamma)
  15. 15. Once the alpha subunit is removed, the enzyme is inactive. When the alpha subunit is bound to GDP, it no longer associates with the enzyme and it exposes binding sites for B/G subunit, so they can all come together.
  16. 16. Signaling Pathways
  17. 17. Alpha subunit
  18. 18. As long as active subunit is bound, the enzyme will continue intracellular signaling.
  19. 19. When bound to GTP, it releases itself from B/G and the activation sites for target proteins/enzymes are now ready to interact
  20. 20. Interacts with target proteins and are activated as long as alpha subunit is bound
  21. 21. Hydrolysis of GTP
  22. 22. GDP makes inactive
  23. 23. How activation takes place
  24. 24. One ligand binds receptor
  25. 25. One h-t-g-p interacts with receptor
  26. 26. One alpha subunit acts with proteins
  27. 27. Enzymes provide an amplification step
  28. 28. Table
  29. 29. Gs heterotimeric G proteins aka alpha s
  30. 30. Stimulates the function of adenylyl cyclase (a.k.a. adenylate cyclase) – designation for alpha subunit of this g protein.
  31. 31. Referred to as alpha-s, when bound to GTP, moves through membrane, attaches to adenylyl cyclase and turns it on. Therefore, that leads to an increase in cAMP in the cell
  32. 32. Adenyly cyclase converts ATP to cAMP
  33. 33. An Active Gs subunit
  34. 34. Alpha subunit is active and bound to GTP
  35. 35. Alpha subunit migrates to adenylyl cyclase and turns on its function
  36. 36. Adenylyl cyclase removes a pyrophosphate (2) from ATP and make cAMP, a favorable reaction by breaking the phospoanhydride bonds holding the Pi on to the ATP
  37. 37. Cyclic form is unstable, being formed to AMP
  38. 38. Necessary so it can be turned off rapidly.
  39. 39. Golf
  40. 40. Olfactory neurons - activates adenylyl cyclase
  41. 41. Alpha subunit is activation subunit
  42. 42. Different protein because it’s in a different cell type.
  43. 43. GI (aka alpha I subunit on Gi protein that carries the function.
  44. 44. Inhibitory/inhibiting – also characterize the alpha subunit as being the
  45. 45. Inhibits adenylyl cyclase therefore decreasing cAMP
  46. 46. In some cells B/G activates K+ channels
  47. 47. Go
  48. 48. Heterotrimeric (as are all) that does different things in different cells, where in some cases the B/G is only active, but sometimes both A and B/G participate in target activation
  49. 49. Beta gamma turns on or opens up K+ channels in the PM allowng K+ to rush out of cell
  50. 50. When A and B/G are both active, Phospholipase c-beta is turned on
  51. 51. PLCB or PLCG both cleave PIP2 (generating DAG that remains attached to membrane and releases IP3 into the cytosol to bind to IP3 receptors in the ER) in the membrane
  52. 52. PLCB activation
  53. 53. HTGprotein called G0 that is turned on after ligand binds to g protein linked receptor – g protein linked receptors
  54. 54. PLCG activation is downstream of enzyme linked receptor.
  55. 55. G1 – transducin
  56. 56. Processes visual information
  57. 57. Structure and Metabolism of cAMP
  58. 58. Can generate kinases
  59. 59. Ligand binds to erceptor
  60. 60. The Roles of G Proteins and Cyclic AMP in Signal Transduction
  61. 61. How cAMP is generated by G proteins
  62. 62. Ligand binds
  63. 63. Activation of heterotrimeric G protein where alpha is bound to GTP
  64. 64. Moves through membrane and interacts with a single molecule of adenylyl cyclase, which is also found in inner leaflet of PM
  65. 65. Activates enzymatic activity
  66. 66. Converts ATP to cAMP
  67. 67. Used for signaling
  68. 68. Rapidly degraded by phosphodiesterase to convert it to AMP
  69. 69. Release energy
  70. 70. When alpha subunit has a GDP attached, the G protein becomes inactive and production of cAMP stops sits in membrane in wait for another signal
  71. 71. What does an increased level of cAMP mean?
  72. 72. cAMP binds to a kinase, called protein Kinase A, which sits in cytosol in inactive state until bound by cAMP. Similar to cGMP. PKG and PKA both have a structure so they have regulatory sites that can be bound by an intracellular signaling protein
  73. 73. when they are bound, open up and release catalytic site that does the phosphorylating.
  74. 74. cGMP, activates PKG, synthesized by guanylyl cyclase
  75. 75. for PKA, two molecules of cAMP must bind to activate
  76. 76. adenylyl cyclase activates cAMP
  77. 77. Once PKA or PKG is activated we have catalytic sites that are turned on ready for phosphorylation, and both target serine and threonine as target proteins.
  78. 78. Recognize the amino acids on either side of the phosphorylated threonine/serine really.
  79. 79. Kinases use particular sequences to indicate a particular site of phosphorylation
  80. 80. When PKA active, bound by cAMP, exposes NLS, and be transported to nucleus, and catalytic site
  81. 81. When in the nucleus, the active PKA will Pi target.
  82. 82. Important target is CREB – cyclic AMP response element binding protein
  83. 83. When phosphorylated by active PKA, regulates gene transcprition by binding to regions on DNA called CRE, or cyclic AMP response element.
  84. 84. CREB binds to CRE, bind to sequence of DNA and geulate gene transcription
  85. 85. In cardiac muscle cells, activation of this pathway increases strength of contraction
  86. 86. In smooth muscle cells, decrease strength of contraction
  87. 87. Neurons – activation of CREB leads to synthesis of neurotrophins, or molecules that help other neurons survive
  88. 88. Liver cells – glycogen synthesis and usage is enhanced by activation of PKA, Pi of CREB and regulation of gene transcription
  89. 89. Important: pathway is the same, but different outcomes.
  90. 90. Can be regulated and adenylyl cyclase can be turned off by alpha subunit binding.
  91. 91. Levels of cAMP by turning on or off adenylyl cyclase
  92. 92. Also for cAMP regulation: active phosphodiesterase will break down cyclic AMP to AMP, and inactivation will cause cAMP to stick around to active more PKA. Synthesis and degredation is regulatable.
  93. 93. Phospholipid signaling
  94. 94. Cleavage of PIP2
  95. 95. Cleaves by phospholipase C Beta or Gamma
  96. 96. PLCB – through g protein activation
  97. 97. PLCG – through enzyme linked receptor
  98. 98. Phospholipase enzyme targets bond between phosphate of inositol ring and oxygen releasing inositiol triphosphate and diacyl glycerol.
  99. 99. IP3 binds to IP3 receptors to release Ca2+ from intracellular storage
  100. 100. DAG and IP3 signaling work together to turn on a kinase in a cell
  101. 101. Activation of these leads to regulation of blood platelets
  102. 102. Smooth muscles
  103. 103. Insulin secretion
  104. 104. Amylase secretion
  105. 105. Glycogen degradation
  106. 106. Antibody production
  107. 107. G protein linked receptor that binds ligand will have a heterotrimeric g proteinactive
  108. 108. Activated alpha will move through membrane and activate PLCB that exposes cleavage site that can cleave phosphoinositol bisphosphosphate into DAG and IP3 signaling molecule
  109. 109. As IP3 moves to receptors on intracellular Ca2+ storage sites, it binds to IP3 receptors and it itself opens up and allow calcium to move to the cytosol. (it is an ion channel, the receptor)
  110. 110. DAG and Ca2+ will activate PKC inside the cell.
  111. 111. PKC enzyme phosphorylates serine and threonine is activated by DAG in phospholipid bilayer, binding of PS (phosphatidalserine, inserted in PM and flipped to inner leaflet) in the lipid bilayer and the binding of calcium that is required for activation of PKC
  112. 112. IP3 signaling pathway is a main way that levels of Ca2+ increase for active PKC
  113. 113. Once active, it can Pi its targets
  114. 114. Importance of Calcium
  115. 115. Uses ATP dependent/independent mechanisms for regulation
  116. 116. Sodium/calcium exchanger at PM for Na into cell used to pump Ca out of cell
  117. 117. CA2+ pump that pumps out calcium out of the cell at all times or into the ER
  118. 118. Ca is in the matrix of mitochondria too
  119. 119. Very local signaling molecule, will happen right on the PM etc
  120. 120. Calmodulin has a complex carboxy and amino termini. Within globular ends, there are two binding sites for calcium so four calcium molecules are bound by calmodulin in cytoplasm and is then activated
  121. 121. Once activated can form kinase, which can wrap itself around a phosphorylating enzyme and stays attached to it. Ca calmodulin complex is PART OF THE KINASE SO IT CANNOT FUNCTION WITHOUT THE ENZYME
  122. 122. CAM Kinasae – calcium calmodulin kinase
  123. 123. Aka Calcium dependendent calmodulin kinase
  124. 124. Can then Pi targets
  125. 125. Calcium binding proteins</li></ul>End lectures<br />Other lecture Notes<br /><ul><li>Protein Kinase A (PKA)
  126. 126. 2 cAMP add to protein kinase A
  127. 127. activated in the same way as protein kinase G
  128. 128. phosphorylates serine and threonine on target substrates.
  129. 129. Active PKA (in nucleus)
  130. 130. Phosphorylates CREB (cycle AMP response element binding protein)
  131. 131. Interacts with sites on the genome that have been called CRE sites. (cAMP response elements)
  132. 132. This interaction drives gene transcription
  133. 133. Skeletal and liver cell, increase glycogen, synthesis and usage
  134. 134. Smooth muscle cells, decrease strength of contraction
  135. 135. Cardiac muscle increases when pathway activated
  136. 136. The cell can monitor the concentration of cAMP by the regulation of phosphodiesterase
  137. 137. Cleavage of PIP2 in the Membrane
  138. 138. Phosphoinositolbisphosphate (PIP2)
  139. 139. Platelet Activation (table slide)
  140. 140. Once PKC isactive, PKC Pi’s targets, and these targets are also serine and threonine sites on target proteins.
  141. 141. Ca2+ is important for intracellular signaling.
  142. 142. Exchangers keep Ca2+ low in cell
  143. 143. Na+/Ca2+ pump
  144. 144. Pumps my hydrolyze ATP to bring Ca2+ out of the cell
  145. 145. Calmodulin is activated by 4 Ca2+
  146. 146. CaM Kinase only active when calmodulin wraps around it, only when it is bound by Ca2+ forming the Ca2+-CaM Kinase